Polyacrylic hydrazides and their applications as glycoprotein reagents

ABSTRACT

A method for detecting a glycoprotein using a solid support is disclosed where the glycoprotein is oxidized by periodate, polyacrylic polyhydrazide which is a copolymer having repeating units possessing a hydrazide group and repeating units possessing hydroxyl groups is coupled to the oxidized glycoprotein and a glycoenzyme or radioactive compound containing aldehyde groups or activated ketone groups is coupled to the polyacrylic polyhydrazide which allows for detection of the glycoprotein. The glycoprotein may be directly attached to the solid support or may be bound to an antigen which is immobilized on the solid support.

FIELD OF THE INVENTION

This invention concerns polymeric hydrazides consisting of an aliphaticC--C backbone and side chains containing hydrazide and other hydrophilicgroups. Such polyhydrazides were found to specifically react with thealdehyde groups generated in the sugar portion of a glycoprotein. Sinceimmunoglobulins are also glycoproteins, the polyhydrazides were used tointroduce a label into the antibody molecule. Therefore, the mostimportant application of the polyhydrazides includes detection ofantibodies of immunoassays and detection of glycoproteins in general.

BACKGROUND OF THE INVENTION

In many research, industrial and clinical laboratories there is agrowing need for indentification of a single protein present in acomplex protein mixture. This is most often achieved by one ortwo-dimensional electrophoretic separation of proteins followed byWestern blotting. Here Western blotting includes transfer of proteins toa suitable membrane, reaction with antibodies specific for oneparticular protein and visualization of the antibodies bound to thatprotein. The visualization of antibodies is done in an indirect way,that is through a signal generated by a label attached to the antibodymolecule. The label is very often an enzyme but other moleculesproducing a suitable signal, such as fluorescent dyes or radioactivecompounds, are also in use. There are several ways to couple a label toan antibody, and the properties of the resulting conjugate are greatlydependent on the coupling method used for its preparation (reference 1).

In addition to detection of a single protein, it is sometimes necessaryto identify a certain class of proteins. This can be achieved through acommon determinant of these proteins. For example, glycoproteins can bedetected through the carbohydrate chains that are covalently linked tothe protein part of the molecule. Thus, the intact carbohydrate chainscan be detected by means of proteins, such as lectins, glycosidases orglycosyltransferases, that recognize those monosaccharides that arepresent in the glycan chains (reference 2). A more general approachinvolves a modification of the carbohydrate chains followed by theirdetection through a group introduced by the modification. It is obviousthat such a group must not be present in an intact protein orglycoprotein molecule. Aldehyde groups are easily and convenientlygenerated in the sugar chains, either chemically or enzymatically, andthen detected by means of a hydrazide reagent and a suitable label(reference 2).

Antibodies, that is immunoglobulins, are also glycoproteins. Therefore,their carbohydrate chains can be used to introduce a label. Thisapproach is very attractive because the carbohydrate chains ofantibodies are apparently not directly involved in the binding of anantigen. Several reports have appeared describing preparation of suchconjugates (references 3 and 4). In one report (references 5) it wasclaimed that an enzyme was coupled to the sugar portion of the antibody,but the procedure described was later found unsatisfactory (reference6). We have found that aldehyde groups introduced into IgG can be usedto cross-like the carbohydrate chains present on each of the two IgGheavy chains (reference 7). The cross-linking reaction by dihydrazidemolecules was much more favorable than the reaction of dihydrazidesthrough only one hydrazide group. This finding has indicated that it isdifficult to introduce free hydrazide groups into IgG by using adihydrazide. As demonstrated in this invention, that can be readilyachieved by polymeric hydrazides which therefore serve as a bridge tothe label.

OBJECTIVES OF THE INVENTION

It is an object of the present invention to provide a process for thepreparation of polyacrylic hydrazides.

It is another object of the present invention to provide a process forcoupling of polyacrylic hydrazides to oxidized glycoproteins in asolution and on a solid phase or support, such as a membrane.

It is another object of the present invention to demonstrate the use ofpolyacrylic hydrazides in detection of glycoproteins in general and ofimmunoglobulins in particular.

It is another object of the present invention to demonstrate detectionof antigens through polyacrylic hydrazides coupled to antibodies.

SUMMARY OF THE INVENTION

We have found that suitable polyacrylic hydrazides are convenientlyprepared through co-polymerization of a neutral, hydrophilic monomer anda monomer containing an activated ester group. Neutral monomers includeacrylamide, N-acryloyl-tris-(hydroxymethyl)-aminomethane andN-acryloyl-2-amino-2-deoxy-D-glucitol, whereas N-acryloxysuccinimide wasused as an activated monomer. The reaction of hydrazine with thecopolymer produced the desired polyacrylic hydrazides. Suchpolyhydrazides couple readily to periodate oxidized glycoproteins, forexample to horse radish peroxidase (HRP) and immunoglobulins. Detectionof glycoproteins on solid support, such as membranes consists mainly ofperiodate oxidation of these glycoproteins and polyhydrazide mediatedcoupling of HRP to the modified glycoproteins. Staining on HRP activitythen locates the glycoproteins. The coupling of HRP is carried outthrough the aldehyde groups generated in the carbohydrate chains of thisglycoenzyme. The detection method provided in this invention is specificfor glycoproteins and its sensitivity for highly glycosylated proteinsis comparable to the sensitivity of Aurodye (Janssen) staining of theblots. Detection of antigens is performed through the antibodies whichhave been oxidized by periodate. The polyhydrazides serve as a bridgebetween the oxidized sugar chains of an antibody and the oxidized sugarchains of an glycoenzyme, such as horse radish peroxidase (HRP). Thisapproach offers several advantages, such as the attachment of a label tothe inert sugar chains of antibodies and the possibility to avoid theuse of secondary antibodies.

In place of the enzyme (HRP) we have also used another label, such as aradioactive compound, in order to improve some properties of the method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are a schematic presentation of the glycoprotein detectionmethod of the present invention showing the steps of reacting aglycoprotein bound to a membrane by periodate, incubating the membranein a solution containing the polyacrylic polyhydrazide, binding anoxidized glycoenzyme to the remaining hydrazide groups and visualizingthe enzyme after incubation with its substrate;

FIGS. 2A-2F show the results of evaluation of different hydrazides fordetection of glycoproteins using the method of the present invention;

FIGS. 3A-3D show glycoprotein detection using the method of the presentinvention with poly(NAGA) polyhydrazide as described in Example 4;

FIGS. 4A-4C show specific detection of glycoproteins afterelectrophoretic separation and transfer to a membrane as described inExample 4 a;

FIGS. 5A-5B show specific detection of glycoproteins according to themethod depicted in FIG. 1 (panel B) and using a polyacrylicpolyhydrazide-peroxidase conjugate (panel A) prepared as described inExample 4 b;

FIG. 6 shows detection of an antigen according to the method of thepresent invention prepared as described in Examples 5 b-f;

FIG. 7 shows another detection of an antigen according to the method ofthe present invention in which all reactions were carried out on amembrane;

FIG. 8 shows comparative results on sensitivity of antigen andglycoprotein detection using the method of the present invention asdescribed in Example 6 b;

FIGS. 9A and 9B show specific detection of glycoproteins with apolyhydrazide labeled with ³² P of a low (panel A) or high (panel B)specific radioactivity; and

FIGS. 10A-10C show specific detection of glycoproteins with differentpolyhydrazides labeled with ¹²⁵ I.

DETAILED DESCRIPTION OF THE INVENTION

Various aspects of the present invention are illustrated by 7 examplesand 8 figures.

EXAMPLE 1 Preparation of polyacrylamide polyhydrazide (PANPH).

The polyhydrazide was obtained frompoly(acrylamide-co-N-acryloxysuccinimide) (PAN) whose synthesis isdescribed in reference 8. Briefly, acrylamide (3.146 g) andN-acryloxysuccinimide (1.48 g) were dissolved in 35 ml of drytetrahydrofurane and polymerized with 20 mg ofazo-bis-(isobutyronitrile) at 55° C. for 24 h. The dried PAN wasdissolved in 20 mM sodium acetate buffer, pH 4.5, at concentration of2%. After cooling to 0° C., the solution was slowly added to ten timesits volume of 1M hydrazide acetate in 0.2M sodium acetate, pH 5.7. Aftertwo hours, the polymeric hydrazide was purified directly by dialysis orit was first precipitated in ethanol. The precipitate was collected bycentrifugation, immediately redissolved on 0.1 m sodium acetate, pH 4.5and dialyzed against the same buffer. The resulting PANPH had ahydrazide content of 0.7-0.9 micromoles per mg dry polymer, asdetermined by trinitrobenzene sulfonic acid with adipic acid dihydrazideas a standard.

EXAMPLE 2 Preparation ofPoly(N-acryloyl-tris-(hydroxymethyl)aminomethane-co-N-acryloxysuccinimide)(PNATN)

In a typical preparation, NAT (2.45 g, 14 mmole) prepared as described(reference 9) and N-acryloxysuccinimide (0.48 g, 2.8 mmole) weredissolved in 35 ml of dry dimethylformamide (DMF) in a 50 ml ampoule anddegassed in vacuo. A stream of dry nitrogen was passed through thesolution for 15 min and then azo-bis-(isobutyronitrile) (AIBN, 33 mg in330 microliters of dry DMF) was added into the ampoule. The ampoule wasquickly sealed and incubated in a water bath at 55° C. for 24 h. Theresulting polymer is purified by precipitation in dry acetone or ethanol(ten times the volume of the polymer solution). The precipitate iscollected by centrifugation and redissolved in 50 mM acetic acid anddialyzed against the same solution. The polymer can be stored at -20° C.or freeze-dried from the acetic acid solution.

b. Preparation ofpoly(N-acryloyl-tris(hydroxymethyl)-aminomethane)-polyhydrazide(PNATPH). PNATPH is prepared by addition of hydrazine to the PNATNpurified as described under 2a or by addition of hydrazine to thepolymer solution in DMF without previous purification of the polymer. Inthe first case, the procedure outlined under example 1 is followed. Inthe second case, at the end of polymerization the ampoule is cooled to-20° C. and carefully opened. A stream of dry nitrogen is immediatelypassed through the solution and anhydrous hydrazine, in five fold molarexcess over N-acryloxysuccinimide and freshly diluted with DMF, isquickly added. The reaction mixture is stirred for about 30 min at 0° C.and then at room temperature. The resulting PNATPH was precipitated inethanol and further treated as described for PANPH (Example 1). When thepolyhydrazide was prepared with NAT and N-acryloxysuccinimide in molarratio of 5:1, the PNATPH had a hydrazide content of 0.3-0.4 micromole/mgdry polymer. PNATPH solution can be stored for several weeks at +4° C.It can be stored for several months frozen at - 20° C. for lyophilizedfrom the sodium acetate buffer.

EXAMPLE 3 Preparation ofpoly(N-acryloyl-2-amino-2-deoxy-D-glucitol)polyhydrazide (PNAGAPH)

In a typical preparation, 0.7 g of N-acryloyl-2-amino-2-deoxy-D-glucitol(N-acryloyl-glucosaminitol, NAGA), prepared as described in reference10, and 0.126 g of N-acryloxysuccinimide were dissolved in 5 ml of dryDMF. After degassing and flushing with nitrogen, 40 μl of AIBN (100mg/ml) was added, the ampoule sealed and heated at 55° C. for 24 h. Asdescribed for PNATPH, the resulting polymer was either purified and thenreacted with hydrazine or hydrazine was immediately added to the DMFsolution of the polymer. In the latter case, the polymer precipitatesbut it can be readily dissolved in the acetate buffer.

EXAMPLE 4 Applications of polyhydrazides coupled to an enzyme indetection of glycoproteins

a. The "two incubations protocol". FIG. 1 shows a scheme for detectionof glycoproteins. A glycoprotein is immobilized on a solid support(membrane) and oxidized by periodate (panel 1). The oxidizedglycoprotein is reacted with a polyhydrazide (panel 2) or anotherbifunction or multifunction hydrazide. Some of the hydrazide groupsreact with the aldehydes generated in the sugar chains of theimmobilized glycoprotein, and some remain free. The free hydrazidegroups are reacted with the aldehyde groups generated in thecarbohydrate chains of a soluble glycoenzyme, such as peroxidase (panel3). The bound enzyme (panel 4), and through it the immobilizedglycoprotein, is located with a suitable substrate which is usuallyconverted to a colored product by the enzyme.

FIG. 2 shows the results of experiments done to evaluate differenthydrazide reagents for detection of glycoproteins. Onto eachnitrocellulose strip, serial dilutions of pre-oxidized proteins (1 mg/mlprotein, 10 mM sodium periodate, 1 h in the dark) were applied. Lane 1always contains fetuin, lane 2 rabbit IgG, lane 3 human IgM and lane 4an unglycosylated protein, E. coli beta-galactosidase, as a control. Theremaining binding sites on all membranes, except that shown in panel A,were blocked by BLOTTO (reference 11). Staining of all proteins in panelA was done by Aurodye as described by the manufacturer. The membranesB-F were then incubated for 45 min with a hydrazide reagent in 0.1Macetate buffer pH 4.5. The membrane in panel B was incubated with 200 mMmalonic acid dihydrazide, in panel C with 200 mM adipic aciddihydrazide, in panel D with 200 mM tricarballilic acid trihydrazide, inpanel E with 0.8 mM PANPH (plus 3% bovine serum albumin) and in panel Fwith 0.8 mM PNATPH. The concentrations of the polyhydrazides are givenas molarities of the hydrazide groups. The membranes were washed (3times with the buffer) and subsequently incubated for 75 min withoxidized horse radish peroxidase at 5-10 μg/ml in 0.1M acetate buffer,containing 0.2M NaCl and 5% nonfat dry milk. The membranes were thenwashed three times with BLOTTO pH 7.5 and stained on peroxidase activitywith 4-chloro-1naphtol (reference 12). The oxidized peroxidase usedabove was prepared by periodate oxidation of the enzyme (5-8 mg/ml in0.1M acetate buffer pH 4.5, 8-10 mM NaIO₄, 2 h at 0° C. in the dark) anddesalting by passing through a PD-10 column (Pharmacia).

As can be seen from FIG. 2 panel 1, practically equal quantities of thefour proteins were applied to the membranes. Of these proteins, onlyhuman IgM is weakly stained with the two dihydrazides (panels B and C).The trihydrazide made possible detection of all glycoproteins (panel D),but IgG was stained very weakly. The polymeric hydrazides (panels E andF) stained strongly all glycoproteins, and a higher sensitivity isachieved by PNATPH (panel F). Under the conditions described, unspecificstaining of the unglycosylated protein, beta-galactosidase in lane 4 ofeach panel, was not observed. The sensitivity of our staining procedureis for some proteins (fetuin and IgM) similar to that of Aurodyestaining (panel A versus panel F).

We have varied many parameters of the glycoprotein staining procedureoutlined above. Thus, polyacrylamide polyhydrazides of different sizeand hydrazide group concentration were prepared or purchased (Sigma).Polyacrylamide polyhydrazides have a general tendency to produce ahigher background and they show a higher unspecific binding tounglycoslyated proteins than PNATPH. This tendency is stronger for thehigh molecular weight polyacrylamide polyhydrazides. Therefore, we haveprepared the PANPH (Example 1), an acrylamide based polymer that isexpected to be of medium size (i.e., below 10 kDa) because the startingproduct (PAN, reference 8) is of that size. PANPH polymers of differentsize were evaluated. We have found that longer PANPH polymers (separatedfrom the shorter ones by ultrafiltration through a PM-30 membrane) givea stronger signal but also a higher background. Accordingly, the longestpolymers producing acceptable background should be used for maximalsensitivity. On the other hand, PNATPH polymers always showed lessbackground staining. We assume that this property of PNATPH is relatedto its more hydrophillic character, coming from the presence of threehydroxyl groups in every repeating unit.

We have also prepared an extremely hydrophilic polyhydrazide, PNAGAPH(Example 3), containing 5 hydroxyl groups in every repeating unit.

FIG. 3 shows the glycoprotein staining done with PNAGAPH. In all panelslane 1 contains fetuin, lane 2 rabbit IgG and lane 3 beta-galactosidase,in concentrations given in FIG. 2. The other conditions are alsoidentical to those described for FIG. 2. Panels A and B were incubatedwith the PNAGAPH prepared from the purified activated polymer, whereaspanels C and D were incubated with the PNAGAPH prepared from theactivated polymer in DMF (see Example 3). Panels A and C were incubatedfor 30 min and panels B and D for 3 h.

The background staining in FIG. 3 is not visible even after a ratherlong incubation (3 h), in contrast to the membranes incubated withPNATPH or PANPH (not shown). From this result we conclude that polymerichydrazides should be as hydrophilic as possible.

In addition to type and size of the polyhydrazides, we have investigatedmany parameters that might have an impact on the performance of theglycoprotein staining procedure. Thus, the concentration of thepolymeric hydrazide was also important. Optimal results were observedwith 0.8 mM hydrazide groups of PANPH and PNATPH. Lower concentrations(below 0.1 mM) give a weaker signal, and higher concentration (5 mM)much higher background.

We have also found that oxidation conditions influence the stainingsensitivity. Thus, at a low periodate concentration the oxidation at pH4.5 results in a much stronger signal than the oxidation at pH 7.5.Incubation time can be lowered by increasing the polyhydrazideconcentration, but the appearance of background was difficult toprevent, except with PNAGAPH. An increase in ionic strength of thePNATPH solution reduces background, but this was not the case withPANPH.

In order to reduce both background and unspecific staining, The PANPHneeds to be incubated in presence of a protein, such as bovine serumalbumin. On the other hand, there is no need for addition of a proteinto PNATPH and PNAGAPH solution, since these two polyhydrazides shownegligible unspecific binding.

The coupling of polyhydrazides to oxidized glycoproteins is convenientlycarried out at pH values from 3.5 to 5.5. Higher values may be used, butthe sensitivity is lower. This is in agreement with the previousfindings (reference 13).

The concentration of oxidized peroxidase can be increased withoutcausing a higher background. The incubation time is then shorter, butmore enzyme is spent. The oxidized peroxidase loses very slowly itsactivity in BLOTTO, 50% in three to four weeks.

We have further used the "two incubations protocol" for detection ofglycoproteins after SDS electrophoretic separation and transfer to apolyvinyldenedifluoride (PVDF) membrane (Millipore). The proteins wereseparated in a 5-20% polyacrylamide gradient gel according to reference14. The transfer of proteins to the membrane was carried out asdescribed in reference 15. The silver staining of the polyacrylamide gelwas done as in reference 16.

FIG. 4 shows a polyacrylamide gel stained with silver (panel A), a PVDFmembrane stained with Aurodye (panel B) and a PVDF membrane stained forglycoproteins according to our "two incubations protocol". The sameproteins are shown in all three gels. In lane s, there are low molecularweight standard proteins from Pharmacia. They include: phosphorylase b(94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonicanhydrase (30 kDa), soybean trypsin inhibitor (20 kDa) and lactalbumin(14.4 kDa). In lanes 2-4 different amounts of the same glycoproteinmixture were applied. The glycoproteins are: invertase (the diffuse bandabove 94 kDa), fetuin (the three bands around 67 kDa), ovalbumin (43kDa) and ribonuclease B (about 22 kDa) containing some ribonuclease A(about 20 kDa). Lane 1 contains 750 ng of each protein, lane 2 250 ng,lane 3 80 ng and lane 4 30 ng.

The membrane C was oxidized with 10 mM NaIO₄ in 0.1M sodium acetatebuffer, pH 4.5 at room temperature for 30 min. It was then incubatedwith PNATPH followed by oxidized peroxidase and staining on peroxidaseactivity, as described for FIG. 2.

FIG. 4 shows that the glycoprotein staining procedure described in thisinvention is characterized by a sensitivity similar to that of silverand Aurodye staining (panel A and B versus panel C). Such a highsensitivity was not achieved by any of the published procedures. Theselectivity of the staining is also remarkable, since of all standardproteins in lane s only the glycoprotein ovalbumin is stained. Inaddition, the nonglycosylated form of ribonuclease (ribonuclease A) isnot stained.

b. The "one incubation protocol".

This procedure differs from the one described in FIG. 1 only in oneimportant detail, that is the steps 2 and 3 are combined into one step.Thus, an oxidized glycoprotein on a solid support is incubated with aglycoenzyme-polyhydrazide conjugate, which is prepared as describebelow.

In general, PANPH or PNATPH are coupled to periodate oxidized HRP. Thecoupling reaction is straightforward (as checked by SDSelectrophoresis), but purification of the conjugate is rathercomplicated because polyhydrazides behave also as chargedmacromolecules. Several examples describing preparation of suchconjugates are given below.

In one experiment, a fraction of PANPH that passed through the PM-30ultrafiltration membrane was used. After coupling the conjugate waspurified from the unbound polyhydrazide by severalconcentration-dilution cycles.

In the second experiment, PANPH was treated with hydrazine in thepresence of a water soluble carbodiimide in order to block all carboxylgroups on the polymer. Since the resulting polymer does not bind to ananionic ion exchanger (Mono Q), the conjugate was bound to a columnpacked with this ion exchanger (10 mM piperazine buffer, pH 10.2) andeluted with 50 mM piperazine-HCl buffer, pH 8.0 containing 0.2M NaCl.

In the third experiment, the purification was attempted over aConcanavalin A-Sepharose column. However, about 90% of the conjugate didnot bind and the portion that bound could be hardly released from thecolumn.

Finally, we have found that purification of the conjugate may not benecessary. When periodate oxidized HRP (2 mM NaIO₄ for 2 h at 0° C.) wasincubated at 1 mg/ml with PNATPH which was 1-4 mM in hydrazide groups,the formed conjugates could be directly used for detection ofglycoproteins.

FIG. 5 shows glycoproteins on a membrane stained according to the "oneincubation protocol" (panel A) and according to the "two incubationsprotocol" (panel B). Lane 1 contains fetuin, lane 2 rabbit IgG and lane3 beta-galactosidase. The incubation conditions were essentially asdescribed for FIG. 2. The conjugate used in the "one incubationprotocol" was not purified.

This figure shows that the sensitivity of the "two incubations protocol"is higher. We have obtained such a result in all instances, regardlesswhether and how the HRP-polyhydrazide conjugate was purified prior toits use in the "one incubation protocol".

EXAMPLE 5 Application of polyhydrazides in detection of antigens

As demonstrated by previous examples, polyhydrazides can act as a bridgethat links the oxidized carbohydrate chains of two glycoproteins. Whenone of them is an antibody and the other an enzyme, the conjugate can beused for detection of an antigen.

The conjugates can be prepared as described by examples a-e and examplef.

a. Goat antibody (against guinea pig immunoglobulins) is oxidized byperiodate (10 mM, 2 h), desalted and incubated overnight with anoxidized peroxidase-PNATPH conjugate (1 mg/ml peroxidase plus 1.5 mMhydrazide). The molar ratio of IgG to HRP was 1:4.

b. The same oxidize antibody was incubated with an oxidizedperoxidase-PNATPH conjugate (1 mg/ml protein plus 3.1 mM hydrazide) with1:4 molar ratio of IgG to HRP.

c. The same oxidized antibody was also incubated with an oxidizedperoxidase--PNATPH conjugate (1 mg/ml protein plus 4.3 mM hydrazide),with 1:4 molar ratio of IgG to HRP.

d. The oxidized antibody was first incubated overnight with PNATPH (0.17μg/ml protein plus 1.1 mM hydrazide) and then with oxidized HRP, with1:4 molar ratio of IgG to HRP.

e. The oxidized antibody was incubated overnight with PNATPH (0.17 μg/mlprotein plus 4 mM hydrazide) and then with oxidized HRP, with 1:4 molarratio of IgG to HRP.

The HRP-PNATPH-IgG conjugates prepared as described under a-e weretested for their ability to detect the antigen.

FIG. 6 shows the result of this experiment. A commercial (Sigma)secondary antibody-HRP conjugate was used in lane 1, the conjugateprepared as described under a in lane 2, under b in lane 3, under c inlane 4, under d in lane 5 and under e in lane 6. In all cases theprimary antibody concentration was 1 μg/ml.

The results clearly show that two of the conjugates we prepared (lane 3and 4) show a similar and one a better sensitivity (lane 2) than thecommercial conjugate. This is a clear demonstration that such conjugatescan be competitive to the conjugates prepared by coupling the enzyme tothe protein part of the antibody molecule.

f. In contrast to examples 5d and 5e, the coupling of an enzyme to anantibody-polyhydrazide conjugate can be done also on a solid support.Thus, oxidized goat anti-guinea pig antibody was coupled to PNATPH (4mM) and then incubated with the membrane containing the antigen. Afterwashing away the unbound IgG--PNATPH, the membrane was incubated withoxidized HRP. The antigen was detected by staining on HRP activity (notshown), but the sensitivity was somewhat lower than shown in FIG. 6,lane 2.

EXAMPLE 6 A novel approach for detection of antigens bound to a solidphase

a. In this approach all reactions are carried out on a solid phase(membrane). First, an antigen is immobilized to a solid support. Second,an oxidized antibody binds to this antigen. Third, a polyhydrazide iscoupled to the sugar part of the antibody molecule. Fourth, an oxidizedglycoenzyme is coupled to the polyhydrazide and the antigen thendetected through a product of the enzyme reaction.

FIG. 7 shows detection of an antigen (guinea pig immunoglobulins)according to the reaction sequence outlined above. The firstnitrocellulose membrane strip was stained with Aurodye. The other threestrips (panel A) were incubated with the oxidized (2 mM sodium peridate,1 h) goat anti-guinea pig antibody at the specified concentrations inBLOTTO pH 7.5 for 1 h. The subsequent incubation with PNATPH andoxidized HRP were done as described in Example 4. Panel B showsdetection of the same antigen with commercial (Sigma) secondary antibodyconjugate. The first antibody concentration was 1 μg/ml and the rabbitanti-goat antibody-HRP conjugate was diluted 1:400 with BLOTTO.

The antigen can be clearly detected by the new approach (FIG. 7), butthe sensitivity is lower than that achieved with the commercialconjugate or demonstrated in Example 5.

b. We assume that the sensitivity of detection of an antigen by the newapproach is limited by the sensitivity of detection of IgG by ourglycoprotein staining procedure. This assumption is supported by theresults shown in FIG. 8.

Panel A and B contained the same four proteins, including fetuin (lane1), rabbit IgG (lane 2), human IgM (lane 3) and beta-galactosidase (lane4). The membrane in panel A was incubated with goat anti-rabbitantibody-HRP (Sigma, diluted 1:200) in BLOTTO pH 7.5 for 2 h. Themembrane in panel B was stained on glycoproteins, as described inExample 4.

FIG. 8 shows that IgG can be detected with about 10 times bettersensitivity by means of the commercial secondary antibody conjugate.However, for another class of antibodies, such as IgM shown in lane 3,the glycoprotein staining is more sensitive than secondary antibodystaining of IgG (lane 2 panel A verses lane 3 panel B). Thus, thisresult indicates that monoclonal IgM antibodies will be detected withbetter sensitivity than IgG. We have also observed that immunoglobulinsIgY from chicken give a stronger signal than mammalian IgG with ourglycoprotein stain. Therefore, the antibodies raised in avian speciesappear well suited for detection of antigens according to the newapproach provided in this invention. Mammalian IgM and chickenimmunoglobulins stain better probably because they contain morecarbohydrate. Hence we expect that the new approach will be particularlywell applicable to the immunoglobulins containing more sugar than IgG.In spite of its currently lower sensitivity when using IgG classantibodies, the new approach is attractive because it offers thefollowing advantages.

The first advantage of the new approach is that a separate preparationof antibody-enzyme conjugates is not necessary.

The second advantage is that there is no foreign molecule (label)introduced into the antibody prior to binding to an antigen. A labelmolecule attached to an antibody often adversely affects binding of thelabeled antibody to its antigen.

The third advantage of this approach is that secondary antibodies arenot necessary. This means that only one animal needs to be sacrificedfor preparation of antibodies against one antigen. Further, falsepositive results and other problems associated with cross-reactions ofsecondary antibodies are avoided.

The fourth advantage is that it is not necessary to purify an antibody.We have found that desalted rabbit serum, containing antibodies againstcreatine kinase, can be used after periodate oxidation to detect theantigen by the procedure outlined above. The amount of periodate usedwas equal to the molar amount of neutral sugars, determined with mannoseas a standard (3 umole/ml sugar was found and the oxidation was donewith 3 mM NaIO₄ for 2 h at 0° C.).

EXAMPLE 7 Application of polyhydrazides labeled with a radioactivecompound in detection of glycoproteins

Radioactive compounds containing a group that reacts with the hydrazidefunction can be coupled to the polyhydrazides. As examples, we describethe preparation of PNATPH labeled with oxidized gamma-³² P-ATP and ¹²⁵ Ilabeled Bolton-Hunter reagent. Polymers of high and low specificradioactivity are prepared.

a. PNATPH of lower specific radioactivity was prepared as follows. To asolution of 5 μl of ³² P-ATP (50 μCi) and 5 μl of 0.2M sodium acetatebuffer, pH 4.5 cooled to 0° C., freshly prepared NaIO₄ (1 μl, 5 mM) wasadded and the mixture incubated for two hours at 0° C. in the dark.Sodium arsenite (1 μl, 5 mM) was then added and the solution was leftfor 30-60 min at room temperature. PNATPH (40 μl, 1.5 mM in hydrazide)was added and the reaction mixture left at +4° C. overnight. A smallcolumn of Sephadex G-25 (approximately 1 ml) was used to purify thelabeled polymer from the unreacted or unoxidized ATP. About 90% of theinitial radioactivity was coupled to the polymer.

b. PNATH of higher specific radioactivity was obtained by lowering theratio of PNATPH to the label. Thus, ³² P-ATP (30 μCi, in 6.5 μl) wasoxidized by periodate (3 mM, 1 μl) and incubated with PNATPH (1 μl, 0.4mM), as described under 5a. After passing of the reaction mixturethrough a small column of Sephadex G-25, over 90% of the initialradioactivity was found in the high molecular weight fraction.

FIG. 9 shows the membranes incubated with ³² P labeled PNATPH of low(panel A) and high (pane B) specific radioactivity. Each membrane wasincubated with 0.9 μCi/ml in sodium acetate buffer for the timesindicated. The proteins were preoxidized and applied to lane 1 (fetuin),lane 2 (rabbit IgG) and lane 3 (beta--galactosidase) in concentrationsgiven in FIG. 2.

It seems that the coupling of radioactive hydrazide to the oxidizedglycoproteins on the membrane is over after 30 min. Longer incubationtime produces only more background. The sensitivity is better with thepolyhydrazide of lower specific radioactivity, probably because theconcentration of the reagent is much higher during the couplingreaction. The overall sensitivity is not better than with theenzyme-labeled polyhydrazides (FIGS. 2-8), but it seems that the signalis more proportional to the amount of glycoproteins.

Polyacrylic hydrazides were also labeled with ¹²⁵ I.

c. A sample of PANPH, (400 μl, 1.4 mM) prepared as described in Example2, was added to 140 μCi of Bolton-Hunter reagent (evaporated from drybenzene) and reacted for 2 h in an ice bath. The solution was thenpassed over a column filled with 7 ml of Amberlite XAD-7 equilibrated insodium acetate buffer pH 4.5. The column was washed with the same bufferand then with 30% methanol in water. About 50% of the appliedradioactivity passed through the column (representing low molecularweight compounds as checked by PD-10 gel filtration) whereas theremaining 50% was eluted with 12 ml of 30% methanol.

FIG. 10 shows the glycoprotein staining through the ¹²⁵I-polyhydrazides. The membrane in panel A was incubated with the labeledPANPH (0.5 μCi/ml) for 2 h and the membrane in panel B was incubatedovernight. The membrane in panel C was incubated with the labeled PNATPHfor 3 h.

These results demonstrate that a long incubation with the labeled PANPHis necessary to detect IgG (lane 2 in panel B versus lane 2 in panel A).The background staining with PANPH is stronger than with PNATPH, andeven after a short incubation (3 h) it is possible to detect IgG withPNATPH (pane C versus panel B). However, the overall sensitivity is notbetter than the sensitivity achieved with the enzyme-labeledpolyhydrazides.

REFERENCES

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We claim:
 1. A method for detection of an oxidized glycoproteincomprising the steps of:a) binding a glycoprotein to a solid support; b)oxidizing the bound glycoprotein with periodate to form an oxidizedglycoprotein wherein aldehyde or ketone groups are formed by oxidizingcarbohydrate residues of said glycoprotein; c) reacting said oxidizedglycoprotein with a polyacrylic polyhydrazide wherein said polyacrylicpolyhydrazide binds to said oxidized glycoprotein, and wherein saidpolyacrylic polyhydrazide is a copolymer comprising repeating unitspossessing a hydrazide group and repeating units possessing hydroxylgroups; d) reacting a label with said polyacrylic polyhydrazide, whereinsaid label contains aldehyde groups or activated ester groups which bindto said polyacrylic polyhydrazide through hydrazide groups on saidpolyacrylic polyhydrazide; and e) detecting said oxidized glycoproteinbound to said support through detection of said polyacrylicpolyhydrazide having said label bound thereto.
 2. The method of claim 1wherein said label is bound to said polyacrylic polyhydrazide beforesaid polyacrylic polyhydrazide is reacted with said oxidizedglycoprotein.
 3. The method of claim 1, wherein said glycoprotein isdirectly bound to said solid support.
 4. The method of claim 3, whereinsaid solid support is a membrane.
 5. The method of claim 3, wherein saidlabel is a glycoenzyme with oxidized sugar chains, and wherein saidoxidized sugar chains of said label are coupled to said oxidizedglycoprotein by means of said polyacrylic polyhydrazide.
 6. The methodof claim 3, wherein said label is a radioactive compound containingaldehyde groups or activated ester groups, and wherein said radioactivecompound is bound to said polyacrylic polyhydrazide through saidaldehyde groups or said activated ester groups.
 7. The method of claim1, wherein said glycoprotein is bound to said solid support throughanother molecule.
 8. The method of claim 7, wherein said solid supportis a membrane.
 9. The method of claim 7, wherein said glycoprotein isbound to said solid support through an antigen.
 10. The method of claim9, wherein said glycoprotein is an antibody.
 11. The method of claim 7,wherein the label is a glycoenzyme with oxidized sugar chains, andwherein the oxidized sugar chains of said label are coupled to theoxidized sugar chains of the oxidized glycoprotein by means of saidpolyacrylic polyhydrazide.
 12. The method of claim 7, wherein said labelis a radioactive compound containing aldehyde groups or activated estergroups, and wherein said radioactive compound is bound to saidpolyacrylic polyhydrazide through said aldehyde groups or said activatedester groups.
 13. A method for detection of an oxidized glycoproteincomprising the steps of:a) oxidizing a glycoprotein with periodate toform an oxidized glycoprotein wherein aldehyde or ketone groups areformed by oxidizing carbohydrate residues of said glycoprotein; b)binding said oxidized glycoprotein to a solid support; c) reacting saidoxidized glycoprotein with a polyacrylic polyhydrazide wherein saidpolyacrylic polyhydrazide binds to said oxidized glycoprotein, andwherein said polyacrylic polyhydrazide is a copolymer comprisingrepeating units possessing a hydrazide group and repeating unitspossessing hydroxyl groups; d) reacting a label with said polyacrylicpolyhydrazide, wherein said label contains aldehyde groups or activatedester groups which bind to said polyacrylic polyhydrazide throughhydrazide groups on said polyacrylic polyhydrazide; and e) detectingsaid oxidized glycoprotein bound to said support through detection ofsaid polyacrylic polyhydrazide having said label bound thereto.
 14. Themethod of claim 13 wherein said label is bound to said polyacrylicpolyhydrazide before said polyacrylic polyhydrazide is reacted with saidoxidized glycoprotein.